C-Glucosidic ellagitannin oligomers from Melaleuca squarrosa Donn ex Sm., Myrtaceae

C-Glucosidic ellagitannin dimers were classified as types A-C according to a putative biogenetic oligomerization mode. They were characterized by different positions of the C-C bond between the phenolic acyl unit in one monomer and the benzylic C-1 of the open-chain glucose core in the other monomer. In recent years, four C-glucosidic tannins, melasquanins A-D (18-21), have been found in the leaves of Melaleuca squarrosa Donn ex Sm. (Myrtaceae). These are characterized as a dimer (melasquanin A) of a dimerization mode (type D), and trimers (melasquanins B-D) based on spectroscopic analysis including various two-dimensional nuclear magnetic resonance (2D NMR) experiments. Melasquanins B (19) and D (21) are C-glucosidic tannin trimers with a structure containing, non-repeating condensation modes, which was hitherto unknown.     

Flavonol glycosides of Warburgia ugandensis leaves

Four new flavonol gycosides: kaempferide 3-O-beta-xylosyl (1-->2)-beta-glucoside, kaempferol 3-O-alpha-rhamnoside-7,4'-di-O-beta-galactoside, kaempferol 3,7,4'-tri-O-beta-glucoside and quercetin 3-O-[alpha-rhamnosyl (1-->6)] [beta-glucosyl (1-->2)]-beta-glucoside-7-O-alpha-rhamnoside, were characterized from a methanolic leaf extract of Warburgia ugandensis. The known flavonols: kaempferol, kaempferol 3-rhamnoside, kaempferol 3-rutinoside, myricetin, quercetin 3-rhamnoside, kaempferol 3-arabinoside, quercetin 3-glucoside, quercetin, kaempferol 3-rhamnoside-4'-galactoside, myricetin 3-galactoside and kaempferol 3-glucoside were also isolated. Structures were established by spectroscopic and chemical methods and by comparison with authentic samples.     

Ericifolin: an eugenol 5-O-galloylglucoside and other phenolics from Melaleuca ericifolia

Ericifolin, an eugenol 5-O-beta-(6'-O-galloylglucopyranoside) possessing the naturally unknown phenolic moiety, 5-hydroxyeugenol, together with the two new phenolics, 2-O-p-hydroxybenzoyl-6-O-galloyl-(alpha/beta)-4C1-glucopyranose and 3-methoxyellagic acid 4-O-rhamnopyranoside have been isolated from the antibacterial leaves extract of Melaleuca ericifolia. In addition, 19 known phenolics were also separated and characterized. All structures were elucidated on the basis of analysis of 1H, 13C NMR, HMQC, HMBC and FTMS spectral data.     

Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase

Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3beta-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1H NMR and CD spectroscopies as (-)-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non-specific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.     

Flavonol tetraglycosides and other constituents from leaves of Styphnolobium japonicum (Leguminosae) and related taxa

Two flavonol tetraglycosides comprising a trisaccharide at C-3 and a monosaccharide at C-7 were isolated from the leaves of Styphnolobium japonicum (L.) Schott and characterised as the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranosides of quercetin and kaempferol. The 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside of kaempferol, the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranosides of kaempferol and quercetin and the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside of kaempferol were also obtained from this species for the first time. Some or all of these flavonol tetra- and triglycosides were detected in 17 of 18 specimens of S. japonicum examined from living and herbarium material, although the most abundant flavonoid in the leaves was generally quercetin 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside (rutin). The triglycosides, but not the tetraglycosides, were detected in herbarium specimens of Styphnolobium burseroides M. Sousa, Rudd & Medrano and Styphnolobium monteviridis M. Sousa & Rudd, but specimens of Styphnolobium affine (Torrey & A. Gray) Walp. contained a different profile of flavonol glycosides. The flavonol tetra- and triglycosides of S. japonicum were also present in leaves of Cladrastis kentukea (Dum. Cours.) Rudd, a representative of a genus placed close to Styphnolobium in current molecular phylogenies. An additional constituent obtained from leaves of Styphnolobium japonicum was identified as the maltol derivative, 3-hydroxy-2-methyl-4H-pyran-4-one 3-O-(4'-O-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-beta-glucopyranoside.     

Flavonol and drimane-type sesquiterpene glycosides of Warburgia stuhlmannii leaves

An investigation of methanolic extract of Warburgia stuhlmannii leaves has led to the isolation of two new drimane-type sesquiterpene glycosides characterized as mukaadial 6-O-beta-D-glucopyranoside, mukaadial 6-O-alpha-L-rhamnopyranoside together with two other novel flavonol glycosides identified as 3',5'-O-dimethylmyricetin 3-O-beta-D-2",3"-diacetylglucopyranoside and 3'-O-methylquercetin 3-O-beta-D-2",3",4"-triacetylglucopyranoside. The known compounds; mukaadial, deacetylugandensolide, quercetin, kaempferol, kaempferol 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-beta-D-glucopyranoside, kaempferol 7-O-beta-D-glucopyranoside, myricetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-sophoroside and isorhamnetin 3-O-beta-D-glucopyranoside were also isolated from the same extract.     

Flavonol and iridoid glycosides of Ajuga remota aerial parts

Six flavonol glycosides characterised as myricetin 3-O-alpha-rhamnosyl-(1'-->2')-alpha-rhamnoside-3'-O-alpha-rhamnoside, 5'-O-methylmyricetin 3-O-[alpha-rhamnosyl (1'-->2')][alpha-rhamnosyl (1'-->4')]-beta -glucoside-3'-O-beta-glucoside, 5'-O-methylmyricetin 3-O-alpha-rhamnosyl (1'-->2')-alpha-rhamnoside 3'-O-beta-galactoside, kaemferol 3-O-rutinoside-7-O-rutinoside, myricetin 3-O-rutinoside-3'-O-alpha-rhamnoside, myricetin 3-O-beta-glucosyl (1'-->2')-beta-glucoside-4'-O-beta-glucoside together with two iridoid glycosides identified as 6,8-diacetylharpagide and 6,8-diacetylharpagide-1-O-beta-(3',4'-di-O-acetylglucoside) have been isolated from extract of Ajuga remota aerial parts. Also isolated from the same extract were known compounds; kaempferol 3-O-alpha-rhamnoside, quercetin 3-O-beta-glucoside, quercetin 3-O-rutinoside, 8-acetylharpagide, ajugarin I and ajugarin II.     

Flavonol pentaglycosides of Cordyla (Leguminosae: Papilionoideae: Swartzieae): distribution and taxonomic implications

A survey of foliar flavonoids in the swartzioid legume genus Cordyla s.l. revealed that three species, C. haraka, C. pinnata and C. richardii, were rich in flavonol pentaglycosides. Their structures were elucidated by spectroscopic and chemical methods as the 3-O-alpha-l-rhamnopyranosyl(1-->3)-alpha-l-rhamnopyranosyl(1-->2)[alpha-l-rhamnopyranosyl(1-->6)]-beta-d-galactopyranoside-7-O-alpha-l-rhamnopyranosides of quercetin and kaempferol (cordylasins A and B, respectively). These compounds were not found in the remaining species, C. africana, C. densiflora, C. madagascariensis (two subspecies) and C. somalensis, which exhibited different profiles of flavonoid glycosides. The distribution of flavonol pentaglycosides in Cordyla s.l. does not support a recent proposal to place both C. haraka and C. madagascariensis in the genus Dupuya [Kirkbride, J.H., 2005. Dupuya, a new genus of Malagasy legumes (Fabaceae). Novon 15, 305-314]. The generic relationship between Cordyla s.l. and Mildbraediodendron is also reassessed on the basis of chemical characters, as the O-linked tetrasaccharide that characterises cordylasins A and B is the same as that found in mildbraedin (kaempferol 3-O-alpha-l-rhamnopyranosyl(1-->3)-alpha-l-rhamnopyranosyl(1-->2)[alpha-l-rhamnopyranosyl(1-->6)]-beta-d-galactopyranoside), the main foliar flavonoid of Mildbraediodendron excelsum. Mildbraedin itself was found to be a minor constituent of leaflet extracts of C. haraka, C. pinnata and C. richardii, and a major constituent of C. somalensis.     

Resistance to Meloidogyne spp. in Allohexaploid Wheat Derived from Triticum turgidum and Aegilops squarrosa

Expression of resistance to Meloidogyne incognita and M. javanica from Aegilops squarrosa was studied in a synthetic allohexaploid produced from Triticum turgidum var. durum cv. Produra and Ae. squarrosa G 3489. The reproductive rate of different races of M. incognita and M. javanica, expressed in eggs per gram of fresh root, was low (P < 0.05) on the synthetic allohexaploid and the resistant parent, Ae. squarrosa G 3489, compared with different bread and durum wheat cultivars. Reproduction of race 2 and race 3 of M. incognita and an isolate of M. javanica was studied on the synthetic allohexaploid and seven cultivars of T. aestivum: Anza, Coker 747, Coker 68-15, Delta Queen, Double Crop, McNair 1813, and Southern Bell. The latter six cultivars are grown in the southeastern United States and reportedly were resistant to M. incognita. Significant differences (P < 0.05) were detected in nematode reproduction on the seven bread wheat cultivars. Reproduction of M. incognita race 3 and M. javanica was highest on Anza. Reproductive rates on the six southeastern United States bread wheat cultivars varied both within and among nematode isolates. The lowest reproductive rates of the three root-knot isolates were detected in the synthetic allohexaploid.     

Volatile oil comparison of cotyledon leaves of chemotypes of Melaleuca alternifolia

Oil quantity and quality were measured for the cotyledon leaves of the commercial terpinen-4-ol chemical variety of Melaleuca alternifolia. Oil yield obtained by ethanolic extraction was 3.8 micrograms per leaf or 2.6% (dry weight basis). The major components of the oil were alpha-pinene (7.4%), beta-pinene (12.0%) and terpinolene (27.3%). The non-commercial terpinolene chemical variety was found to be rich in 1,8-cineole (12.5%) and terpinolene (25.4%). The non-commercial 1,8-cineole chemical variety was rich in 1,8-cineole (37%) with significant quantities of alpha-pinene (15.5%), beta-pinene (23.3%) and terpinolene (10.9%). The cotyledon leaf composition, when compared with that of mature leaf from the same chemical variety, was found to be biased toward pinene and terpinolene biogenetic pathway constituents and hence not a good indicator of mature tree quality especially for the commercial terpinen-4-ol chemical variety. The implications of these analyses for the determination of M. alternifolia plantation quality and the understanding of oil formation, are discussed.     

Monoterpenoid accumulation in Melaleuca alternifolia seedlings

Individual leaves of the commercial terpinen-4-ol type of Melaleuca alternifolia were examined both quantitatively and qualitatively for volatile constituents from the emergence of the first true leaves, through to 6-week-old tenth leaf set material. A GC internal standard addition method was used to measure changes in oil composition and the accumulation of volatile constituents expressed on a dry weight, unit leaf area and whole leaf basis. In the early stages of seedling growth, leaves contained higher concentrations of terpinolene, alpha-pinene and beta-pinene and lower concentrations of terpinen-4-ol, sabinene and cis-sabinene hydrate than mature leaf. Concentrations of the former constituents fell and the latter rose by the time leaf set 10 was 6 weeks old. Key constituent, 1,8-cineole remained in similar concentration throughout ontogeny. The variation in concentration of other key constituents during early stages of seedling development suggests that caution is required in extrapolating seedling leaf data to mature tree oil quality.     

Flavonol tetraglycosides from fruits of Styphnolobium japonicum (Leguminosae) and the authentication of Fructus Sophorae and Flos Sophorae

The dried fruits and seeds of Styphnolobium japonicum (L.) Schott (syn. Sophora japonica L.) are used in traditional Chinese medicine and known as Fructus Sophorae or Huai Jiao. The major flavonoids in these fruits and seeds were studied by LC-MS and other spectroscopic techniques to aid the chemical authentication of Fructus Sophorae. Among the flavonoids were two previously unreported kaempferol glycosides: kaempferol 3-O-β-glucopyranosyl(1 → 2)-β-galactopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-xylopyranosyl(1 → 3)-α-rhamnopyranosyl(1 → 6)[β-glucopyranosyl(1 → 2)]-β-glucopyranoside, the structures of which were determined by NMR. Two further tetraglycosides were identified for the first time in S. japonicum as kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-glucopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside; the latter was the main flavonoid in mature seeds. The chromatographic profiles of 27 recorded flavonoids were relatively consistent among fruits of similar ages collected from five trees of S. japonicum, and those of maturing unripe and ripe fruits were similar to a market sample of Fructus Sophorae, and thus provide useful markers for authentication of this herbal ingredient. The flower buds (Huai Mi) and flowers (Huai Hua) of S. japonicum (collectively Flos Sophorae) contained rutin as the main flavonoid and lacked the flavone glycosides that were present in flower buds and flowers of Sophora flavescens Ait., reported to be occasional substitutes for Flos Sophorae. The single major flavonoid in fruits of S. flavescens was determined as 3′-hydroxydaidzein.     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.     

Two ellagitannins from Punica granatum heartwood

In the course of studies on polyphenol metabolism in Punica granatum heartwood two new ellagitannins, diellagic acid rhamnosyl (1-->4) glucopyranoside and 5-O-galloylpunicacortein D were isolated and characterized together with four known tannin metabolites, punicacortein D, punicalin, punicalagin and 2-O-galloylpunicalin. Structures of the isolated compounds were established by chromatography, chemical degradation, UV and 1D/ 2D 1H/13C NMR spectroscopy.     

Triumphalone, a diketone from the volatile oil of the leaves of Melaleuca triumphalis, and its spontaneous conversion into isotriumphalone

The major component (35-65%) of the volatile oil obtained by steam distillation of the leaves of Melaleuca triumphalis has been identified as (rel)-1beta-pentyl-1alpha,6alpha-dihydroxy-3,3,5,5-tetramethylcyclohexa-2,4-dione (trivial name triumphalone). Relative stereochemistry was established by nuclear Overhauser experiments and X-ray studies on the 2-(3,5-dinitrobenzoic acid) derivative. The remainder of the oil was composed of mono- and sesquiterpene hydrocarbons and alcohols. On prolonged standing the presence of a rearrangement product of triumphalone was observed which was characterized as (rel)-1beta-pentyl-1alpha,3alpha-dihydroxy-4,4,6,6-tetramethylcyclohexa-2,5-dione (trivial name isotriumphalone), presumably arising from an acid catalyzed shift of the pentyl group from C-1 to C-2.     

Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia

Chemical study of Cunonia macrophylla, a New Caledonian Cunoniaceae, based on bioactive effects of a crude methanol extract of the leaves, detected bioactive tannins for the first time in this plant family. These ellagitannins have been identified as ellagic acid-4-O-beta-D-xylopyranoside (6), mallorepanin (3), mallotinic acid (1) along with corilagin (2), chebulagic acid (4), ellagic acid (5) and gallic acid (7) and have been shown to possess antimicrobial activity and to inhibit xanthine oxidase. Antimicrobial effects on bacterial human pathogens (Staphylococcus aureus, Corynebacterium accolans) and on a plant pathogen (Erwinia carotovora) as well as on a human pathogenic yeast (Candida albicans) were investigated. Activity is reported here for the first time for compounds 1, 3, 4 and 6. The inhibitory effects of all molecules against xanthine oxidase in relation to their structure was evaluated and compared. Compound 6 presented the best activity and seems to be of considerable interest for further studies.     

Flavonoid triglycosides from the seeds of Syzygium aromaticum

Two new apigenin triglycosides, apigenin 6-C-[beta-D-xylopyranosyl-(1'-->2')-beta-D-galactopyranoside]-7-O-beta-D-glucopyranoside and apigenin 6-C-[beta-D-xylopyranosyl-(1'-->2')-beta-D-galactopyranoside]-7-O-beta-D-(6-O-p-coumarylglucopyranoside) were isolated from the ethanol extract of the seeds of Syzygium aromaticum. Their structures were elucidated by chemical and spectral analysis (UV, FABMS, 1H, 13C NMR, HMQC, HMBC, NOESY and DEPT).